Growth, Differentiation and Sexuality

132 N.L. Glass and A. Fleissner
during mating occur in a stepwise fashion and
involve a series of signaling modules, the Rho- and
Ras-type GTPases, which regulate the polarization
of the actin cytoskeleton and the concomitant
polarization of the secretory apparatus to the point
of growth (Nelson 2003). In S. cerevisiae,acomplex
between Gβγ and the Cdc42 activating protein,
Cdc24, localizes to the tip of the mating projection.
Activation of Cdc42 is associated with alterations
in cytoskeletal organization and polarization
involved in chemotropic interactions (Nern and
Arkowitz 1998; for review, see Etienne-Manneville
2004). In the basidiomycete Ustilago maydis, the
yeast-like haploid sporidia respond to pheromone
secreted by a compatible partner, by forming
conjugation tubes; myosin-V (myo5) mutants were
defective in this process (Weber et al. 2003). In S.
cerevisiae, the myosin-V protein, Myo2, is involved
in vesicle delivery along actin cables and is required
for polarized secretion (Karpova et al. 2000).
In filamentous fungi, hyphal tip growth is also
associated with highly polarized and localized
exocytosis. Unlike S. cerevisiae, however, where secretion
and polarization are primarily actin-based,
a functional microtubule cytoskeleton is required
for the transport of vesicles to the hyphal tip and
for hyphal tip growth in filamentous ascomycete
species. Disruption of microtubule function or
its motors affects hyphal tip growth (Seiler et al.
1997). Within the cell, the transport and fusion of
vesicles associated with secretion is accomplished
by integral membrane proteins, termed SNAREs
(soluble N-ethyl-maleimide-sensitive factor attachment
protein receptor; Ungar and Hughson
2003). In N. crassa, transformants containing
silenced copies of putative plasma membranelocalized
SNARES showed reduced growth, hyphal
diameter and conidiation. The defects in nsyn1
and nsyn2 mutants are consistent with differential
impaired vesicle fusion in hyphal tips (Gupta and
Heath 2002; Gupta et al. 2003).
In N. crassa, a number of temperature-sensitive
mutants have recently been identified that show
altered hyphal morphology and branching at restrictive
temperatures (Seiler and Plamann 2003).
The genes mutated in these morphological mutants
were identified via complementation and includemanythatencodepolaritycomponents,such
as the homolog of the Rho-type GTPase, CDC42,
and the guanine exchange factor (GEF) for Cdc42,
CDC24. A second class of morphological mutants
has mutations in components of the secretory apparatus;
the pleiotropic phenotypes may be asso-
ciated with defects in intracellular vesicle trafficking,
which is required for hyphal tip growth. Although
the growth and branching phenotypes of
these mutants have been described, a systematic
microscope survey of the effect of these mutations
on self-anastomosis has not been done. In addition,
a number of filamentous fungal-specific genes
or genes not previously suspected of having polarization
defects in other organisms, when mutated,
give rise to strains affected in polarization (Osherov
et al. 2000; Gatherar et al. 2004; Pearson et al.
2004). These data suggest that some aspects of tip
growth, polarization and, most probably, anastomosis
require functions/genes that are specific to
filamentous fungi.
C. Contact, Adhesion and Cell Wall Breakdown
Hyphal fusion is a temporally and spatially regulated
process that involves degradation of the cell
wall, followed by plasma membrane fusion. From
the time of cell wall contact between fusion hyphae
to membrane contact, two steps are required: adhesion
of fusion hyphae, and subsequent degradation
of the intervening cell wall. Via live cell imaging of
the hyphal fusion process, the Spitzenkörper was
observed to be associated with the site of the future
pore (Hickey et al. 2002; Fig. 7.3B). These observations
suggest that the Spitzenkörper plays a role
in vesicle trafficking involved in the secretion of
extracellular adhesives and cell wall-degrading enzymes.
Upon physical contact, the hyphae (or hypha/peg)
involved in fusion often switch from polar
to non-polar growth, resulting in a swelling of the
hyphae/hypha at the fusion point (Fig. 7.3B). The
delivery of vesicles containing cell wall-degrading
enzymes to the point of fusion may be analogous
to the role the Spitzenkörper plays during hyphal
tip growth, in which highly polarized exocytosis of
vesicles at hyphal apices is an essential requirement
(Grove and Bracker 1970; Howard 1981; Bartnicki-
Garcia et al. 1995).
Extracellular electron-dense material associated
with hyphal fusion events has been observed
in C. parasitica (Newhouse and MacDonald 1991),
which may be involved in adhesion of participating
hyphae. Leakage of cytoplasm during anastomosis
is not observed, suggesting that hyphae are tightly
adhered to each other (Hickey et al. 2002). To complete
the fusion process, the intervening cell wall
between the two fusion hyphae must be dismantled.
These observations indicate that a switch in